The invention relates to an immunochemical method for detecting the deposit of Bacillus thuringiensis delta-endotoxin or pesticidally-active fragment thereof on a plant or tree. The invention further relates to kits for using such a method.
Bacillus thuringiensis is the most widely used biopesticide. Bacillus thuringiensis is a motile, rod-shaped, gram-positive bacterium that is extensively distributed in nature, especially in soil and insect-rich environments. During sporulation, Bacillus thuringiensis produces a parasporal crystal inclusion(s) which is insecticidal upon ingestion to suceptible insect larvae of the orders Lepidoptera, Diptera, and Coleoptera. The inclusions may vary in shape, number, and composition. They are comprised of one or more proteins called delta-endotoxins, which may range in size from 27-140 kDa. The insecticidal delta-endotoxins are generally converted by proteases in the larval gut into smaller (truncated) toxic polypeptides, causing midgut destruction, and ultimately, death of the insect (Hxc3x6fte and Whiteley, 1989, Microbiological Reviews 53:242-255).
There are several Bacillus thuringiensis strains that are used as bioinsecticides in the forestry, agricultural, and public health areas. Bacillus thuringiensis subsp. kurstaki and Bacillus thuringiensis subsp. aizawai produce delta-endotoxins specific for Lepidoptera. A delta-endotoxin specific for Coleoptera is produced by Bacillus thuringiensis subsp. tenebrionis (Krieg et al., 1988, U.S. Pat. No. 4,766,203). Furthermore, Bacillus thuringiensis subsp. israelensis produces delta-endotoxins specific for Diptera (Goldberg, 1979, U.S. Pat. No. 4,166,112).
The delta-endotoxins are encoded by cry (crystal protein) genes which are generally located on plasmids. The cry genes have been divided into six classes and several subclasses based on relative amino acid homology and pesticidal specificity. The major classes are Lepidoptera-specific (cryI); Lepidoptera-and Diptera-specific (cryII); Coleoptera-specific (cryIII); Diptera-specific (cryIV) (Hxc3x6fte and Whiteley, 1989, Microbiological Reviews 53:242-255); Coleoptera- and Lepidoptera-specific (referred to as cryV genes by Tailor et al., 1992, Molecular Microbiology 6:1211-1217); and Nematode-specific (referred to as cryV and cryVI genes by Feitelson et al., 1992, Bio/Technology 10:271-275).
Delta-endotoxins have been produced by recombinant DNA methods. The delta-endotoxins produced by recombinant DNA methods may or may not be in crystal form.
The deposition of a delta-endotoxin onto a plant or tree by application, particularly by aerial application, is complicated by a number of factors including canopy architecture of the plants or trees, meteorological conditions, dilution of the delta-endotoxin formulation, and atomization of the delta-endotoxin formulation during application. In forestry, it is estimated that the deposit efficiency is in the range of 10-50% of the emitted volume in the application of Bacillus thuringiensis delta-endotoxin formulations.
A specific problem in the art is to assess directly the extent of coverage or deposit of a delta-endotoxin after the delta-endotoxin is applied to a plant or tree to control a destructive pest. This assessment is very important for preventing the pesticidal destruction of a plant or tree by alerting the applicator that further application of the delta-endotoxin is needed.
The art has had a long felt, but unfulfilled need for a method that would allow the direct measurement of a delta-endotoxin deposited on a plant or tree by being able to take samples of leaves from plants in a field or from trees in a forest, and directly measuring the deposit of the delta-endotoxin on the leaf, as well as delta-endotoxin deposited on tree bark. Fulfillment of this need would be very advantageous in the art since it would allow the direct determination of the extent of coverage of a delta-endotoxin, and, furthermore, provide an indication of the need for follow-up applications in areas not sufficiently covered to prevent destruction by a pest.
Generally, the activity of Bacillus thuringiensis delta-endotoxin is determined by bioassay. Specifically, the delta-endotoxin is incubated with its target pest, and the increase in mortality and/or stunting of growth of the insect is determined. However, there are a number of disadvantages to bioassays. Bioassay is a labor intensive, time consuming process with a low capacity for sample throughput for quantitative analyses. It requires the rearing of the target species and maintaining a constant colony which is healthy and will perform consistently in the assays. Additionally, since insects are biological organisms, they are prone to the variability that accompanies the use of biological organisms in an assay system xe2x80x94+/xe2x88x9220%. These disadvantages preclude the use of bioassay in assessing the deposit of a Bacillus thuringiensis delta-endotoxin.
A dye incorporated into the pesticidal formulation prior to application can be used as an indirect marker for determining deposition. However, the use of a dye marker for determining deposit is limited in that it can be used only under experimental test conditions and for relatively small application areas. Furthermore, spray cards for measuring the deposit are used which requires significant effort in placing the cards prior to application and in analyzing the cards following application. Dye incorporation is, therefore, not a practical way for determining deposit.
In the prior art, polyclonal and monoclonal antibodies have been generated that specifically react with delta-endotoxin. Polyclonal antibodies have been obtained to the delta-endotoxins of a number of subspecies of Bacillus thuringiensis (Krywienzzcyk, 1977, Publication 1P-X-16, Insect Pathology Research Institute, Canadian Forest Service, Sault Sainte Marie, Ontario, Canada). Monoclonal antibodies have been obtained to the delta-endotoxin of Bacillus thuringiensis subsp. kurstaki (Huber-Lukac et al., 1986, Infection and Immunity 54:228-232; Groat et al., in Analytical Chemistry of Bacillus thuringiensis, ACS Symposium Series 432, Leslie A. Hickle and William L. Fitch, eds., 1990, pp. 88-97), Bacillus thuringiensis subsp. thuringiensis (Huber-Lukac et al., 1982, Experentia 38:1103-1105), Bacillus thuringiensis subsp. berliner (Hxc3x6fte et al., 1988, Appl. Environ. Microbiol. 54:2010-2017) and Bacillus thuringiensis subsp. israelensis (U.S. Pat. No. 4,945,057). However, a practical and reliable method for assessing deposit of a Bacillus thuringiensis delta-endotoxin has not resulted from the availability of these antibodies.
It is an object of the present invention to provide an immunochemical method and kits thereof for assessing directly the deposition of a Bacillus thuringiensis delta-endotoxin after the delta-endotoxin is applied to a plant or tree for controlling a pest.
The present invention is directed to an immunochemical method that satisfies the need to directly measure the deposition of a Bacillus thuringiensis delta-endotoxin or pesticidally-active fragment thereof on a plant or tree. Said method comprises (a) isolating the delta-endotoxin from said sample; (b) reacting the isolated delta-endotoxin of step (a) with at least one antibody or Fab1, F(abxe2x80x2)2, or Fvfragment thereof, in which said antibody binds specifically to the delta-endotoxin; and (c) observing the presence or absence of binding of the antibody of step (b) to said delta-endotoxin. The amount of delta-endotoxin present on the sample may be determined by comparing the amount of binding of the Bacillus thuringiensis delta-endotoxin in the sample to the antibody of step (b) to the amount of binding of a known amount of Bacillus thuringiensis delta-endotoxin to said antibody.
In a specific embodiment, the sample is reacted with two antibodies. In one embodiment, one antibody is a polyclonal antibody specific to a Bacillus thuringiensis delta-endotoxin or pesticidally-active fragment thereof. In another embodiment, the one antibody is a monoclonal antibody specific to a Bacillus thuringiensis delta-endotoxin or pesticidally-active fragment thereof. In another embodiment, the other antibody is a polyclonal antibody specific to a Bacillus thuringiensis delta-endotoxin or pesticidally-active fragment thereof. In another embodiment, the other antibody is a monoclonal antibody specific to a Bacillus thuringiensis delta-endotoxin or pesticidally-active fragment thereof. In a preferred embodiment, both antibodies are polyclonal antibodies specific to the delta-endotoxin or pesticidally-active fragment thereof from Bacillus thuringiensis subsp. kurstaki. 
The invention is also directed to a kit for detecting a Bacillus thuringiensis delta-endotoxin or pesticidally-active fragment thereof deposited on a sample from a plant or tree. Such a kit comprises (a) an extraction buffer for isolating the delta-endotoxin from said sample; and (b)at least one antibody or Fab1, F(abxe2x80x2)2, or Fv fragment thereof, in which said antibody binds specifically to the delta-endotoxin. In one embodiment, the antibody may be attached to a solid support. The kit may also comprise a second antibody which is labeled with a reporter molecule. Furthermore, the kit may also comprise a standard delta-endotoxin or pesticidally-active fragment thereof of known amount.